Project Summary The development and application of novel chemical tools can provide fundamental insights into biomedical processes. The research program outlined in this proposal focuses on three core areas: 1) the development and application of chemosensors for phospho-regulatory enzymes, 2) new methodologies for constructing designer signaling networks, and 3) fundamental studies of protein misfolding. Protein phosphorylation plays a central role in cellular signaling. Consequently, the enzymes that maintain the phosphoproteome, protein kinases and protein phosphatases, are considered key drug targets in human disease. Currently, indirect proxies are used to estimate activity perturbations of kinases and phosphatases associated with disease development. Although useful, these proxies do not provide a direct measure of enzymatic activity, leading to inaccurate estimates of kinase and phosphatase activity. As a result, a clear understanding of the role of kinase and phosphatase activity perturbations during the development and progression of human disease is lacking. Therefore, there is a critical need for the development and application of chemical tools to directly quantify kinase and phosphatase activity in human disease states. In this proposal, we will leverage a phosphorylation-sensitive amino acid to construct a panel of kinase and phosphatase activity probes and apply this panel to develop longitudinal activity profiles of signaling changes during human disease progression. The finely tuned activity of signaling proteins is essential for normal physiology. Indeed, perturbations in the activity of signaling proteins are central to the development of human disease. Unfortunately, the field still lacks a unified approach to modulating the activity of multiple signaling proteins simultaneously in living systems in order to model human disease. To address this critical need, we will leverage split-protein reassembly in order to define a lexicon of standardized parts for fine-tuning the activity of signaling nodes in living cells. In the long-term, this set of mix-and-match parts will be utilized to model human disease states and identify potential drug targets. Lastly, our laboratory will investigate the fundamental aspects of protein misfolding, which is now recognized as a central pathological mechanism in numerous human disease states. The current lack of approaches to assess protein misfolding and aggregation in living systems has created a critical need for the development of novel methodologies to address this gap. By leveraging a novel, luminescence-based assay for protein misfolding and aggregation our laboratory will assess the molecular determinants of protein aggregation in living cells. In addition, we will utilize this approach to identify molecules capable interfering with protein aggregation.